Health Relatedness: A central question in neurodegenerative diseases is how specific populations of neurons undergo cell death. Selective degeneration of motor neurons occurs in the setting of diverse insults including RNA viruses such as HIV and West Nile, and in heritable mutations of ubiquitously expressed genes such as SOD1. It is unclear why proteins that are ubiquitously expressed, or why viruses that are not specifically neurotropic, should render motor neurons selectively vulnerable to death. The selective vulnerability of motor neurons may relate to their unique metabolic demands or, alternatively, they may possess a repertoire of proteins that contextually favor cell-death pathways. We have identified expression of TLR3, an innate immune pattern recognition receptor for duplex RNA, on multiple neuronal populations. While TLR3 is expressed in all neurons examined, activation on cortical neurons or sensory neurons has no effect on cell survival. By contrast, activation of TLR3 results in selective loss of motor neurons in primary dissociated spinal cord cultures. Administration of a TLR3 agonist in neonatal mice results in a loss of islet-1 positive neurons within the ventral horns. A clue into the cause of selective motor neuron death may be the observation that TLR3 recruits either cell-death activating or repressing proteins and we found its activation induces cleaved caspase-3 in motor neurons exclusively. Long-term goals: Our long-term goal is to define the function of TLR3 as a potential mediator of motor neuron death in translational models of motor neuron disease. Our hypothesis is that activation of the TLR3 pathway induces selective death in motor neurons as a consequence of their unique vulnerability. Research Approach: In aim 1, we will further investigate the selective vulnerability of motor neurons to TLR3 mediated death by expanding on our existing preliminary data and studying the cellular mechanisms involved. Different neuronal populations will be assessed for cell death following TLR3 activation in comparison to motor neurons. We will then determine if TLR3 functions autonomously in motor neurons to cause death or if TLR3 functions in an additional glial cell type to cause motor neuron death using glial- neuronal co-cultures. We will also determine if TLR3 functions in vivo, in both neonatal and adult animals, to induce selective death of motor neurons by comparing TLR3 knock-outs with strain matched controls. In aim 2 we will study the molecular basis by which TLR3 mediates selective death of motor neurons. We will determine if the proximal adaptor protein TRIF is necessary for TLR3 mediated death of motor neurons using cell culture and in vivo models. We will examine the essential branch points of TLR3 signaling to determine if the balance of NF-kB and caspase-8 activation favors cell death in motor neurons and cell survival in other neuronal types. Since both viral and native cellular ligands exist for TLR3, this pathway represents a new and unique link between infection, cell injury, and motor neuron disease. PUBLIC HEALTH RELEVANCE: Selective motor injury occurs in a number of neurodegenerative diseases such as Primary Lateral Sclerosis, pure Hereditary Spastic Paraplegia, Kennedy's disease, Spinal Muscular Atrophy, Progressive Muscular Atrophy, and amyotrophic lateral sclerosis, as well as in viral diseases such as Polio, HIV, and West Nile. Why specific populations of motor neurons are selectively vulnerable to the genetic and environmental conditions associated with these diseases is unknown. We have identified a receptor termed TLR3 present on all neurons, that when activated, induces death to only motor neurons. This application pursues research to define the relevance of this finding to diseases of the motor neuron.